The present invention relates principally to the production of vanillin (4-hydroxy-3-methoxybenzaldehyde), particularly to the production of vanillin other than by extraction from the Vanilla pod.
Vanillin is an important food and drink flavouring agent and a major flavour component of natural vanilla from the Vanilla pod. The use of natural vanilla is limited by its high price. Synthetic vanillin, commonly derived from sulphite liquors produced during the processing of wood pulp for paper manufacture, is frequently used as a low-cost vanilla substitute. Alternative biological processes for the production of natural vanillin and allied flavourings would have considerable industrial value and utility, most particularly if such processes could facilitate the production of vanillin and/or allied flavourings directly in a fermented food or beverage.
The mechanism of vanillin biosynthesis in Vanilla remains substantially uncharacterised. M. H. Zenk (Anal. Z. Pflanzenphysiol 53, 404-414 (1965)) showed that vanillin was derived from trans-ferulate (4-hydroxy-3-methoxy-trans-cinnamate) and proposed a mechanism analogous to the classical xcex2-oxidation of fatty acids, with cleavage of a xcex2-keto thioester to produce acetyl SCoA and vanilloyl SCoA (4hydroxy-3-methoxybenzoyl SCoA) and subsequent reduction and CoASH release to generate vanillin. C. Funk and P. E. Brodelius (Plant Physiol. 94, 95-101; 102-108 (1990); 99, 256-262 (1992)), proposed a different route, in which the 4-hydroxy group of trans-ferulate became successively methylated and demethylated during the pathway of vanillin biosynthesis; however, the detailed enzymology was not elucidated. In potato tubers and in the fungus, Polyporus hispidus (C. J. French, C. P. Vance and G. H. N. Towers, Phytochemistry 15, 564-566 (1976)), in cell cultures of Lithospermum erythrorhizon (K. Yazaki, L. Heide and M. Tabata, Phytochemistry 30, 2233-2236 (1991)) and in cell cultures of carrot (J.-P. Schnitzler, J. Madlung, A. Rose and H. U. Seitz, Planta 188, 594-600 (1992)), evidence was obtained from in vitro studies that the corresponding analogue of vanillin, 4-hydroxybenzaldehyde, was an intermediate in the formation of 4-hydroxybenzoate from 4-coumarate (4-hydroxy-trans-cinnamate). There was no requirement for ATP or CoASH, thus apparently ruling out a xcex2-oxidation mechanism. Further studies with cell-free extracts of Lithospermum erythrorhizon, however, have in contrast recently established the presence of a xcex2-oxidation route for the conversion of 4-coumarate to 4-hydroxybenzoate (R. Lxc3x6scher and L. Heide, Plant Physiol. 106, 271-279 (1994)); in this case, the conversion was dependent on ATP, Mg2+ ions and NAD+ and proceeded via 4-hydroxybenzoyl SCoA, without the intermediate formation of 4-hydroxybenzaldehyde.
In the Gram-negative bacterium, Pseudomonas acidovorans, trans-ferulate was shown to be catabolised to vanillate and acetate, apparently via vanillin (A. Toms and J. M. Wood, Biochemistry 9, 337-343 (1970)). Although in cell-free extracts NAD+ was necessary for the oxidation of vanillin to vanillate and for the further oxidation of vanillate to protocatechuate and formate, no mention was made of any other cofactor requirements. Further studies of ferulate utilisation in Pseudomonas species have been reported (V. Andreoni and G. Bestetti, FEMS Microbiology Ecology 53, 129-132 (1988); T. Omori, K. Hatakeyama and T. Kodama, Appl. Microbiol. Biotechnol. 29, 497-500 (1988); Z. Huang, L. Dostal and J. P. N. Rosazza, Appl. Env. Microbiol. 59, 2244-2250 (1993)); however, these have not sought to elucidate further the mechanism of the two-carbon cleavage of ferulate. Zenk et al (1980) Anal. Biochem. 101, 182-187 describe a procedure for the enzymatic synthesis and isolation of cinnamoyl-CoA thioesters using a bacterial system. In contrast, the enzymology and genetics of the utilisation of simple benzene derivatives, including benzoic acids and phenols, by Pseudomonas have been intensively studied (T. K. Kirk, T. Higuchi and H.-M. Chang (eds.), xe2x80x9cLignin biodegradationxe2x80x9d, CRC Press, Boca Raton, Fla, USA (1980); D. T. Gibson (ed.), xe2x80x9cMicrobial degradation of organic compoundsxe2x80x9d, Marcel Dekker, New York (1984); J. L. Ramos, A. Wasserfallen, K. Rose and K. N. Timmis, Science 235, 593-596 (1987); C. S. Harwood, N. N. Nichols, M. K. Kim, J. L. Ditty and R. E. Parales, J. Bacteriol. 176, 6479-6488 (1994); S. Romerosteiner, R. E. Parales, C. S. Harwood and J. E. Houghton, J. Bacteriol. 176, 5771-5779 (1994); J. Inoue, J. P. Shaw, M. Rekik and S. Harayama, J. Bacteriol. 177, 1196-1201 (1995)).
A survey of potential microbial routes to aromatic aldehydes, including routes (i) from trans-cinnamic acids, (ii) from benzoic acids by reduction and (iii) by conversion of aromatic amino acids to phenylpyruvic acids followed by treatment with base, has been presented by J. Casey and R. Dobb (Enzyme Microb. Technol. 14, 739-747 (1992)).
U.S. Pat. No. 5,128,253 describes a method of producing vanillin from ferulic acid by various microorganisms and extracts thereof or enzymes derived therefrom in the presence of a sulphydryl compound but does not disclose what any of the enzymes involved in the conversion of ferulic acid to vanillin are. U.S. Pat. No. 5,279,950 is a continuation-in-part application of U.S. Pat. No. 5,128,253 which additionally describes that Vanilla calluses can be used in the process.
WO 94/13614 describes the production of vanillin from ferulic acid by the action of Vanilla root material and makes use of an adsorbent, such as charcoal, to extract vanillin but does not disclose the specific enzymes involved.
EP 0 453 368 describes that a culture of Pycnoporus can convert trans-ferulic acid into vanillin but does not disclose the specific enzymes involved.
WO 94/02621 describes the production of vanillin from trans-ferulic acid by the action of a lipoxygenase enzyme. EP 0 405 197 describes the production of vanillin from eugenol/isoeugenol by bacteria from the genera Serratia, Klebsiella and Enterobacter by oxidation.
Vanillin may also be produced from phenolic stilbenes as is mentioned in Hagedorn and Kaphammer (1994) Ann. Rev. Microbiol. 48, 773-800.
Vanillic acid is also a useful compound as it can be polymerised into oligomers or used as a monomer in the synthesis of polyesters; similarly p-hydroxybenzoic acid is also useful for polymer synthesis.
A first aspect of the invention provides a method of producing vanillin comprising the steps of
(1) providing trans-ferulic acid or a salt thereof; and
(2) providing trans-ferulate:CoASH ligase activity (enzyme activity I), trans-feruloyl ScoA hydratase activity (enzyme activity II), and 4-hydroxy-3-methoxyphenyl-xcex2-hydroxy-propionyl SCoA (HMPHP SCoA) cleavage activity (enzyme activity III).
The advantages of the present invention over chemical synthesis or extraction from the Vanilla pod include (i) economic advantage over extraction from Vanilla pod and freedom from geographical dependence on Vanilla growing areas; (ii) the ability to produce vanillin by a natural process, involving biological catalysts; (iii) the benefits of generating a natural flavour in situ in a fermented food or beverage, if the genes are expressed in appropriate food-grade hostsxe2x80x94eg lactic acid bacteria or yeasts; and (iv) the possibility of expanding the range of plants in which vanillin and related substances might be produced and from which they might be extracted. These and other examples of the methods of the invention are described in more detail below.
We have determined the mechanism of chain-shortening of trans-ferulate (trans-ferulic acid) by a strain of Pseudomonas fluorescens (named Ps. fluorescens biovar. V, strain AN103 and which we have abbreviated at some points to AN103) isolated from soil. Our data indicate clearly that vanillin (4-hydroxy-3-methoxy benzaldehyde) is an intermediate and that the mechanism does not involve xcex2-oxidation. The vanillin pathway of Ps. fluorescens biovar. V, strain AN103 is described in FIG. 1. Trans-ferulic acid (or a salt thereof) is interconverted with trans-feruloyl ScoA in the presence of CoASH; trans-feruloyl SCoA is interconverted with 4-hydroxy-3-methoxyphenyl-xcex2-hydroxypropionyl SCoA (HMPHP SCoA); and HMPHP SCoA is interconverted with vanillin.
For convenience, trans-ferulate:CoASH ligase activity is called enzyme activity I, trans-feruloyl SCoA hydratase activity is called enzyme activity II; and HMPHP SCoA cleavage activity is called enzyme activity III. The interconversions performed by these enzyme activities is shown in FIG. 1.
The method of producing vanillin provided by the invention therefore includes the steps of exposing trans-ferulic acid or a salt thereof to enzyme activity I and forming a product, exposing the said product of enzyme activity I to enzyme activity II to form a product and exposing the said product of enzyme activity II to enzyme activity III to form a product.
Trans-ferulic acid or a salt thereof may be provided directly, for example by supplying pre-prepared trans-ferulic acid or a salt thereof, or it may be provided indirectly, for example by supplying a precursor of trans-ferulic acid or a precursor of a salt of trans-ferulic acid and means to convert the said precursor into trans-ferulic acid or a salt thereof. As is described in more detail below, it is convenient if the precursor is an ester of trans-ferulic acid and the means to convert said ester is a suitable esterase. By xe2x80x9cproviding trans-ferulate:CoASH ligase activity (enzyme activity I), trans-feruloyl ScoA hydratase activity (enzyme activity II), and 4-hydroxy-3-methoxyphenyl-xcex2-hydroxy-propionyl SCoA (HMPHP ScoA) cleavage activity (enzyme activity III)xe2x80x9d we include the provision of the enzyme activities in any suitable form to effect the said production of vanillin. For example, as is discussed in more detail below, the method of the invention specifically includes, but is not limited to, the provision of the enzyme activities (a) by intact or permeabilised Ps. fluorescens biovar. V, strain AN103 or a mutant thereof, (b) at least one of enzyme activities II or III of which is in a form substantially free of cellular material, (c) by intact or permeabilised cells in culture, particularly microorganisms, which have been genetically modified to contain genes which encode enzyme activities II or III (for example, food grade microorganisms such as lactic acid bacteria and brewing yeast), and (d) by plants which have been genetically modified to contain genes which encode said enzyme activities.
It is preferred if means for converting vanillin to a non-vanillin product is absent or reduced. Of course, the enzyme activity III is not such a means. Conveniently, these enzyme activities are provided by the soil bacterium Pseudomonas fluorescens biovar. V, strain AN103 the said bacterium being that deposited under the Budapest Treaty at the National Collection of Industrial and Marine Bacteria Limited, AURIS Business Centre, 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland under Accession No NCIMB 40783, or a mutant or variant thereof. By xe2x80x9cmutant or variant thereofxe2x80x9d we include any mutant or variant of the said bacterium provided that the bacterium retains the said enzyme activities whether or not at the same levels. It will be appreciated that the said enzyme activities can be retained even if the genes encoding said enzymes are mutated. For example, mutants which constitutively express (as opposed to conditionally or inducibly express) the said enzyme activities are particularly useful mutants of Ps. fluorescens biovar. V, strain AN103, as are variants in which one or more of the enzymes with the said activities exhibit more favourable kinetic characteristics (for example, an increased turnover number or a decreased Km)
When Ps. fluorescens biovar. V, strain AN103 is growing on trans-ferulate it will derive maximum benefit if vanillin is catabolised further in order to provide more energy. However, in order to maximise the production of vanillin by Ps. fluorescens biovar. V, strain AN103 it is preferred that the means for converting vanillin to a non-vanillin product is absent or reduced. We have found that in Ps. fluorescens biovar. V, strain AN103 vanillin is converted to vanillic acid or a salt thereof by vanillin:NAD+ oxidoreductase. It is preferred if a mutant of Ps. fluorescens biovar. V, strain AN103 wherein the vanillin:NAD+ oxidoreductase activity is absent or reduced is used in the method. Such a mutant can be made using a gene replacement strategy with a disrupted vanillin:NAD oxidoreductase gene, or a sequence of DNA from which this gene has been deleted. Gene replacement is well known in the art of bacterial genetics. Alternatively, isolation of such a mutant may be achieved by classical chemical mutagenesis, selecting on the basis of inability to grow on vanillin.
It will be appreciated that there are other means for converting vanillin to a non-vanillin product and it is preferred if these are absent or reduced in the method.
Although Ps. fluorescens biovar. V, strain AN103 or mutants or variants thereof themselves are useful in the method of the invention as whole cells or permeabilised or immobilised cells, it is preferred if the enzyme activities I, II and III are provided by an intact-cell-free system of Ps. fluorescens biovar, V, stain AN103 or a mutant or variant thereof. Suitable systems and extracts may be used by methods well known in the art, for example by French pressure cell or sonication followed by centrifugation. Alternatively, whole cells may be permeabilised using methods well known in the art, for example using detergents such as dimethyl sulphoxide (DMSO).
Using such an intact-cell-free system allows the necessary substrates and any cofactors to reach readily the relevant enzymes and for the products to be released readily into the reaction medium if this is necessary for further reaction; however as discussed below, at least some of the enzymes of the invention may be involved in substrate (metabolic) channelling.
We have found that none of the enzyme activities I, II and III from Ps. fluorescens biovar. V, strain AN103 is dependent on NAD+ whereas enzyme activity IV from Ps. fluorescens (vanillin:NAD+ oxido-reductase) requires NAD+.
Thus, a preferred way of reducing means for converting vanillin to a non-vanillin product in an intact-cell-free system of Ps. fluorescens biovar. V, strain AN103 (or in a cell-permeabilised system of Ps. fluorescens biovar. V, strain AN103) is to omit NAD+ from the reaction system. Any exogenous NAD+ is readily and rapidly depleted by the presence of trans-ferulate in the system.
For the microorganisms of the present invention which can be used in the method of vanillin production, including Ps. fluorescens biovar. V, strain AN103, at least three main types of bioreactor may be used for the biotransformation reactions: the batch tank, the packed bed and the continuous-flow stirred tank; their applications and characteristics have been reviewed (M. D. Lilly in xe2x80x9cRecent Advances in Biotechnologyxe2x80x9d, eds. F. Vardar-Sukan and S. S. Sukan, Kluwer Academic Publishers, Dordrecht, 1992, pp 47-68 and loc. cit.).
As is described in more detail below, enzyme activities II and III are available free from other enzyme activities, for example directly or indirectly from Ps. fluorescens biovar. V, strain AN103 and from other organisms or cells which have been genetically modified to express genes encoding the said enzyme activities.
It will be appreciated that other microorganisms will be found which will be useful in the methods of the invention, for example, by screening. Such microorganisms and methods of screening and methods of use form part of the invention. The method of screening for other microorganisms possessing activities I, II and III is essentially that already described in the Materials and Methods section in the Examples for the isolation of AN 103. The important aspect is isolation from an environment rich in trans-ferulate or related compounds (eg 4-trans-coumarate, trans-caffeate [3,4-dihydroxy-trans-cinnamate] which, as described below, may also be substrates for enzyme activity I) and selection for growth on trans-ferulate (preferably) as sole carbon source. In practice, preferred sources are those in which plant-derived materials are being degraded; in addition to soil or compost, this would include the outflow or residues from factories or other installations processing such materialsxe2x80x94eg sugar-beet factories, cocoa fermentation heaps etcxe2x80x94and the contents of the gastro-intestinal tract, particularly of ruminants and other herbivores. It is possible that anaerobes might be found possessing these activities and due account can readily be taken of this in the isolation procedure. A priori, isolation of organisms with these activities might also be possible from marine environments.
Genera in which further microorganisms useful in the invention will be found include Pseudomonas, Arthrobacter, Alcaligenes, Acinetobacter, Flavobacterium, Agrobacterium, Rhizobium, Streptomyces, Saccharomyces, Penicillium and Aspergillus.
An alternative or additional approach is to use any one of the Pseudomonas genes encoding enzyme activities II or III described herein or redundant sequences designed from the Pseudomonas enzyme amino-acid sequences in DNA probes or PCR amplification strategies to find related genes in other organisms. As is made more clear below, enzymes and nucleotide sequences which are functionally equivalent to those of isolated from AN103 but which differ in sequence form part of the invention.
Our studies indicate that the enzyme which interconverts trans-ferulate and trans-feruloyl SCoA in Ps. fluorescens biovar. V, strain AN103 uses Coenzyme A (CoASH), ATP and Mg2+ or other functionally equivalent cofactors. Thus, it is preferred that the method further comprises the step of (3) providing any one of the cofactors CoASH, ATP or Mg2+ or other functionally equivalent cofactors. ATP is adenosine triphosphate. It is well known that other functionally equivalent cofactors can substitute in some cases for CoASH, ATP or Mg2+. For example Mn2+ may be used in place of Mg2+ and derivatives or analogues of ATP, preferably with a hydrolysable xcex3-phosphate, may be used in place of ATP.
We have also determined that, at least when the enzyme activity I is provided by the Pseudomonas AN103 enzyme which interconverts trans-ferulate and trans-feruloyl SCoA and which enzyme uses ATP and Coenzyme ASH, it is convenient to include a system wherein either one, or both, of the cofactors Coenzyme ASH and ATP is recycled. The following ATP generation and CoASH recycling systems are preferred.
ATP generation:
(i) trans-Ferulate+CoASH+ATP+H2Oxe2x86x92Vanillin+Acetyl SCoA+AMP+PPi (overall reaction catalysed by Ps. fluorescens biovar. V, strain AN103 extract)
(ii) AMP+ATP⇄2 ADP (adenylate kinase)
(iii) Acetylxcx9cP+ADP⇄Acetate+ATP (acetate kinase)
(iv) Sum: trans-Ferulate+CoASH+2 Acetylxcx9cP+H2Oxe2x86x92Vanillin+Acetyl SCoA+2 Acetate+PPi
CoASH recycling is achievable using commercially-available citrate synthase (EC 4.1.3.7) and citrate lyase (EC 4.1.3.6), viz:
(v) Acetyl SCoA+Oxaloacetate+H2O⇄Citrate+CoASH (citrate synthase)
(vi) Citrate⇄Oxaloacetate+Acetate (citrate lyase)
Overall sum, (iv)-(vi):
(vii) trans-Ferulate+2 Acetylxcx9cP+2 H2Oxe2x86x92Vanillin+3 Acetate+PPi
The acetylxcx9cP used in the overall process, (vii), would not itself be generated by enzymic means; however, none of its atoms would appear in the vanillin product.
Acetyl phosphate is commercially available or can be synthesised using the method described by Stadtman (1957) Meth. Enzymol. 3, 228-231.
The reagents are commercially available from, for example Sigma Chemical Co, Fancy Road, Poole, Dorset, UK. Citrate lyase is typically from Enterobacter aerogenes; citrate synthase is typically from chicken heart, pigeon breast muscle or porcine heart.
Thus, it is preferred if coenzyme ASH is recycled using the enzymes citrate synthase and citrate lyase; and it is preferred if the ATP is generated using the enzymes adenylate kinase (EC 2.7.4.3) and acetate kinase (EC 2.7.2.1).
The co-factor recycling system is particularly preferred when using an intact-cell-free system.
Trans-ferulic acid or a salt thereof is readily available from plant material. Suitably, trans-ferulic acid or a salt thereof is released from the plant material by the action of ferulic acid esterase. Thus, in a particularly preferred embodiment of the invention the trans-ferulic acid or salt thereof is provided by the action of ferulic acid esterase on plant material.
Trans-ferulic acid and trans4-coumaric acid can together represent up to 1.5% by weight of the cell walls of temperate grasses (R. D. Hartley and E. C. Jones, Phytochemistry 16, 1531-1534 (1977)). Trans-ferulic acid is reported to comprise 0.5% (w/w) of wheat bran (M. C. Ralet, J.-F. Thibault and G. Della Valle, J. Cereal Sci. 11, 249-259 (1990)), 3.1% of maize bran (L. Saulnier, C. Marot, E. Chanliaud and J.-F. Thibault, Carbohydr. Polym. 26, 279-287 (1995)) and 0.8% of sugar beet pulp (V. Micard, G. M. G. C. Renard and J.-F. Thibault, Lebensm.-Wiss. u-Technol. 27, 59-66 (1994)). These materials are amongst the preferred sources of trans-ferulic acid. Since trans-ferulic acid is present esterified with cell-wall polysaccharides, hydrolysis is essential. Alkaline or acid hydrolysis is possible, but enzymic hydrolysis is preferred. Typically, the initial step is the partial enzymic hydrolysis of carbohydrates (arabinans, xylans, rhamnogalacturanans) to which trans-ferulate is linked, followed by the release of trans-ferulate from the oligosaccharide fragments by trans-ferulic acid esterase activity. In practice, both steps may occur simultaneously in the reaction mixture. Descriptions of representative laboratory-scale processes are available in the literature (for example see L. P. Christov and B. A. Prior, Enzyme Microb. Technol. 15, 460-475 (1993)); C. B. Faulds and G. Williamson, Appl. Microbiol. Biotechnol. 43, 1082-1087 (1995); C. B. Faulds, P. A. Kroon, L. Saulnier, J.-F. Thibault and G. Williamson, Carbohydrate Polymers 27, 187-190 (1995)). Phenolic acid-releasing enzymes have been reported from a number of microorganisms, including Streptomyces olivochromogenes (C. B. Faulds and G. Williamson, J. Gen. Microbiol. 137, 2337-2345 (1991)), Penicillium pinophilum (A. Castanares, S. I. McCrae and T. M. Wood, Enzyme Microb. Technol. 14, 875-884 (1992)), Neocallimastix spp. (W. S. Borneman, R. D. Hartley, W. H. Morrison, D. E. Akin and L. G. Ljungdahl, Appl. Microbiol. Biotechnol. 33, 345-35,1 (1990)), Schizophyllum commune (R. C. MacKenzie and D. Bilous, Appl. Envir. Microbiol. 54, 1170-1173 (1988)) and Aspergillus spp. (M. Tenkanen, J. Schuseil, J. Puls and K. Poutanen, J. Biotechnol. 18, 69-84 (1991); C. B. Faulds and G. Williamson, Microbiology 140, 779-787 (1994)). A trans-ferulic acid esterase (XYLD) has been characterised from Pseudomonas fluorescens subsp. cellulosa, together with an arabinofuranosidase (XYLC) and an endoxylanase (XYLB; L. M. A. Ferreira, T. M. Wood, G. Williamson, C. B. Faulds, G. P. Hazlewood and H. J. Gilbert, Biochem. J. 294, 349-355 (1993)). The genes for all three enzymes have been isolated (G. P. Hazlewood and H. J. Gilbert, in xe2x80x9cXylans and Xylanasesxe2x80x9d, eds. J. Visser, G. Beldman, M. A. Kusters-van Someren and A. G. J. Voragen, Elsevier, Amsterdam, pp 259-273 (1992)). All of these references are incorporated herein by reference.
Thus, advantageously the trans-ferulic acid or a salt thereof may be provided by the action of trans-ferulic acid esterase on said ester. More particularly, it is advantageous to introduce a gene encoding said esterase into a host cell or organism which is being used in the methods of the invention. Thus, it is convenient to introduce a trans-ferulic acid esterase gene, such as the aforementioned XYLD gene, into a plant which is being used in the methods of the invention.
Although, as described above, the method may be performed using enzyme activities I, II and III which are provided by Ps. fluorescens biovar. V, strain AN103 or mutants or variants thereof themselves, or intact-cell-free extracts thereof, it is preferred if at least one of the enzyme activities II and III is provided by a substantially purified enzyme. Substantially purified enzymes with enzyme activities II and III are described below.
In a particularly preferred embodiment of the invention the method of the first aspect of the invention further comprises providing a compound, in addition to trans-ferulic acid or a salt thereof, which may be converted by any one of enzyme activities I, II or III into a desirable product. Suitably said compound is converted by any one or more of said enzyme activities into a product which is found in, and preferably contributes to the taste or aroma of, vanilla as extracted from Vanilla pod.
Vanilla as extracted from Vanilla pod contains vanillin as a major component but also smaller quantities of desirable components such as p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid. Typically these components, and vanillin, are present as glucosides in green vanilla pods as well as in the free form. However, upon hydrolysis or fermentation of the green pods or of hydrolysis of the fermented pods, most of the components are present in the free form.
Thus, it is particularly preferred if said compound is any one of trans-4-coumaric acid or a salt thereof, trans-4-coumaroyl SCoA, trans-caffeic acid or a salt thereof, trans-caffeoyl SCoA, or 3,4-methylenedioxy-trans-cinnamic acid or a salt thereof. By the action of one or more of enzyme activities I, II or III trans4-coumaric acid or a salt thereof and trans-4-coumaroyl SCoA are converted to p-hydroxybenzaldehyde; trans-caffeic acid or a salt thereof and trans-caffeoyl SCoA are converted to 3,4-dihydroxybenzaldehyde; and 3,4methylenedioxy-trans-cinnamic acid or a salt thereof is converted to heliotropin.
It is preferred if the compound is trans4-coumaric acid or a salt thereof or trans-4-coumaroyl SCoA and that the desirable product is 4-hydroxybenzaldehyde which is a significant component of natural Vanilla extract.
The enzyme activities I, II and III from Ps. fluorescens biovar V, strain AN103 are able to use trans-caffeate and trans-4-coumarate, (and, as appropriate, the products of their reaction with enzyme activity I) with reasonable efficiency whereas cinnamate and 3,4-methylenedioxy-trans-cinnamate, although may be used as substrates, are poor substrates of the AN103 enzymes.
Thus, the method of the first aspect of the invention is suited to make vanilla flavourings and aromas which more closely resemble the vanilla from Vanilla pod.
The method of the first aspect of the invention may, in certain circumstances, also be performed using the host cells and genetically modified cells and organisms as described below in more detail.
A second aspect of the invention provides a method of producing vanillic acid, or a salt thereof, comprising the steps of
(1) providing trans-ferulic acid or a salt thereof;
(2) providing trans-ferulate:CoASH ligase activity, trans-feruloyl SCoA hydratase activity, and 4-hydroxy-3-methoxyphenyl-xcex2-hydroxy-propionyl SCoA (HMPHP SCoA) cleavage activity; and
(3) providing an activity that interconverts vanillin and vanillic acid.
For convenience, the activity that interconverts vanillin and vanillic acid is called enzyme activity IV. Conveniently the activity is provided by vanillin:NAD+ oxidoreductase (vanillin dehydrogenase). Suitably, this activity is provided by Ps. fluorescens biovar. V, strain AN103. Methods of converting vanillin to vanillic acid or a salt thereof are also known in the art, for example Perestelo et al (1989) App. Environ. Microbiol. 55, 1660-1662 describes the production of vanillic acid from vanillin by resting cells of Serratia marcescens and Pomelto and Crawford (1983) App. Environ. Microbiol. 45, 1582-1585 describe whole-cell bioconversion of vanillin to vanillic acid by Streptomyces viridosporus. 
The method of producing vanillic acid provided by the invention therefore includes the steps of exposing trans-ferulic acid or a salt thereof to enzyme activity I and forming a product, exposing the said product of enzyme activity I to enzyme activity II to form a product, exposing the said product of enzyme activity II to enzyme activity III to form a product, and exposing the said product of enzyme activity III to enzyme activity IV to form a product.
It will be appreciated that vanillic acid can be made by the same means as vanillin is made in the method of the first aspect of the invention provided, of course, that enzyme activity IV is supplied.
A further preferred embodiment of the first aspect of the invention comprises the further step of separating vanillin from the other reaction components.
Vanillin, and other aromatic aldehydes, are, for example, recoverable by extraction with solvent, including supercritical carbon dioxide, and by organophilic pervaporation, using membranes constructed of hydrophobic polymers (G. Bengston and K. W. Bodekker, in xe2x80x9cBioflavour 95xe2x80x9d, eds. P. Étixc3xa9vant and P. Schreier, INRA, Paris, pp 393-403 (1995); S. M. Zhang and E. Drioli, Separ. Sci. Technol. 8, 1-31 (1994)); pervaporation technology has been applied, for example, to the recovery of flavour compounds of wine (N. Rajagopalan and M. Cheryan, J. Membrane Sci. 104, 243-250 (1995)). Solid-phase extraction, followed by desorption with solvent, is also possible, though less preferred.
However, in some circumstances, particularly where minor reaction products are present which are similar to compounds present in the vanilla isolated from Vanilla pod, vanillin is not isolated.
A further preferred embodiment of the second aspect of the invention comprises the further step of separating vanillic acid or a salt thereof from the other reaction components.
Vanillic acid and other carboxylic acids may, for example, be recovered by solid-phase extraction, by solvent extraction under acidic conditions, or by pertraction; for example, L. Boyadzhiev and I. Atanassova (Process Biochemistry 29, 237-243 (1994)) describe the recovery of the aromatic amino acid, phenylalanine, by pertraction.
A third aspect of the invention provides Pseudomonas fluorescens biovar. V, strain AN103 as deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Limited, AURIS Business Centre, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland under Accession No NCIMB 40783, or a mutant or variant thereof. Preferred mutants and variants are the same as those preferred in the first aspect of the invention. A particularly preferred mutant of Ps. fluorescens biovar. V, strain AN103 is one which accumulates vanillin when provided with trans-ferulic acid or a salt thereof. Conveniently, this is a mutant of Ps. fluorescens biovar. V, strain AN103 wherein vanillin:NAD+ oxidoreductase activity is absent or reduced. Suitably, there is a mutation in the gene encoding vanillin:NAD+ oxidoreductase such that the enzyme activity is absent or substantially reduced. Such a mutant can be made as described above.
A fourth aspect of the invention provides a polypeptide which, in the presence of appropriate cofactors if any, is capable of catalysing the interconversion of trans-feruloyl SCoA and 4-hydroxy-3-methoxy-phenyl-xcex2-hydroxypropionyl SCoA (HMPHP SCoA). Such a polypeptide has enzyme activity II. Conveniently, the polypeptide comprises trans-feruloyl SCoA hydratase; more conveniently the polypeptide comprises trans-feruloyl SCoA hydratase from Ps. fluorescens biovar. V, strain AN103 or fragments or variants thereof which have at least 1% of the specific activity of the native enzyme (in relation to trans-feruloyl SCoA hydratase activity), preferably at least 10%, more preferably at least 100%.
The enzyme activity is readily purified as described in the Examples. Modifications to this procedure may be readily made by the person skilled in the art so that a polypeptide with enzyme activity II can be obtained from any suitable source making use of the enzyme activity II assay procedure described in the Examples.
It is preferred if the polypeptide of the fourth aspect of the invention comprises the amino acid sequence.
or a fragment or variant thereof.
The amino acid sequence is that given in FIG. 12 as that encoded by nucleotides 2872 to 3699.
A fifth aspect of the invention provides a polypeptide which, in the presence of appropriate cofactors if any, is capable of catalysing the interconversion of 4-hydroxy-3-methoxyphenyl-xcex2-hydroxy-propionyl SCoA (HMPHP SCoA) and vanillin. Such a polypeptide has enzyme activity III. Conveniently, the polypeptide comprises HMPHP SCoA cleavage enzyme; more conveniently the polypeptide comprises HMPHP SCoA cleavage enzyme from Ps. fluorescens biovar. V, strain AN103 or fragments or variants thereof which have at least 1% of the specific activity of the native enzyme (in relation to HMPHP SCoA cleavage activity), preferably at least 10%, more preferably at least 100%.
The enzyme activity is readily purified as described in the Examples. Modifications to this procedure may be readily made by the person skilled in the art so that a polypeptide with enzyme activity III can be obtained from any suitable source-making use of the enzyme activity III assay procedure described in the Examples.
It is preferred if the polypeptide of the fifth aspect of the invention comprises the amino acid sequence
or a fragment or variant thereof.
The amino acid sequence is that given in FIG. 12 as that encoded by nucleotides 2872 to 3699.
A sixth aspect of the invention provides a polypeptide comprising the amino acid sequence
or a fragment or variant thereof.
The amino acid sequence is that given in FIG. 12 as that encoded by nucleotides 3804 to 5249.
As described in detail in the examples, this polypeptide sequence encodes an enzyme with vanillin:NAD+ oxidoreductase activity from Ps. fluorescens biovar V., strain AN103.
By xe2x80x9cvariantsxe2x80x9d we include deletions, insertions and substitutions either conservative or non-conservative, where such changes may reduce or enhance the activity, or may not substantially alter the activity. In particular, the seventh aspect of the invention includes the complete polypeptide sequence of Ps. fluorescens biovar V, strain AN103 vanillin:NAD+ oxidoreductase and this polypeptide itself.
By xe2x80x9cconservative substitutionsxe2x80x9d is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using the methods of protein engineering and site-directed mutagenesis as described below and as is well known in the art.
A preferred embodiment of the invention is a polypeptide as defined in the fourth, fifth or sixth of the invention which is substantially pure.
By xe2x80x9csubstantially purexe2x80x9d we mean that the polypeptide is substantially free of other polypeptides, or other macromolecules, with which it is usually found in nature. Suitably, the polypeptide is substantially free of any other polypeptides or macromolecules. It is preferred if the polypeptide has less than 50% by weight of any other polypeptide, preferably less than 10%, more preferably less than 1%, still more preferably less than 0.1% and most preferably less than 0.01%.
Polypeptides can be purified using methods known in the art. It is preferred if the polypeptide is the product of a recombinant DNA.
A single polypeptide chain may comprise more than one of the enzyme activities (II) trans-feruloyl SCoA hydratase activity or (III) HMPHP SCoA cleavage activity.
Our data in the Examples shows that, in the case of Ps. fluorescens biovar. V, strain AN103, enzyme activities II and III are found in the same polypeptide chain, the sequence of which is given as the preferred polypeptides of the fifth and sixth aspects of the invention. Thus, when enzyme activities II and III are provided in any aspect of the invention it is most convenient if they are provided in the same polypeptide chain.
It will be appreciated that, using protein engineering methods or chemical cross-linking it may be possible to produce a single molecule which has enzyme activities II and III. Such a molecule, therefore, forms a further aspect of the invention.
An seventh aspect of the invention provides a polynucleotide encoding a polypeptide as defined in any one of the fourth, fifth or sixth aspects of the invention.
By xe2x80x9cpolynucleotidexe2x80x9d we include RNA and DNA. DNA is preferred.
Thus, this aspect of the invention provides a polynucleotide which encodes any one of a polypeptide which has (II) trans-feruloyl SCoA hydratase activity; (III) HMPHP SCoA cleavage activity; (IV) vanillin:NAD+ oxidoreductase activity; or a polypeptide which has more than one of these activities. Preferably the polynucleotide is derived from Ps. fluorescens biovar. V, strain AN103. A preferred polynucleotide comprises all or at least a part of the Ps. fluorescens DNA contained within the cosmid clone pFI793 as deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Limited, AURIS Business Centre, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland under Accession No NCIMB 40777, or a fragment or variant thereof.
The isolation of the cosmid clone pFI 793 is described in Example 5; pFI 793 includes DNA which encodes polypeptides which have enzyme activities II, III and IV. The cosmid clone pFI 793 itself, the genes contained in the Ps. fluorescens DNA thereof, and variants thereof form separate aspects of the invention.
A variant of a polynucleotide includes any insertion, deletion or substitution of the sequence which encodes a fragment or variant of a polypeptide as defined above.
For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, Strategies and Applications of In Vitro Mutagenesis, Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) of the invention can be used to obtain other DNA sequences that hybridise with it under conditions of high stringency. Such DNA includes any genomic DNA.
Accordingly, the polynucleotide of the invention includes DNA that shows at least 55 percent, preferably 60 percent, and most preferably 70 percent homology with the polynucleotide sequences identified in the invention, provided that such homologous DNA encodes a protein which is usable in the methods described herein.
DNAxe2x80x94DNA, DNA-RNA and RNAxe2x80x94RNA hybridisation may be, performed in aqueous solution containing between 0.1xc3x97SSC and 6xc3x97SSC and at temperatures of between 55xc2x0 C. and 70xc2x0 C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By high stringency we mean 2xc3x97SSC and 65xc2x0 C. 1xc3x97SSC is 0.15M NaCl/0.015M sodium citrate.
xe2x80x9cVariantsxe2x80x9d of the polynucleotide include polynucleotides in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 50% and preferably at least 90 or 95%) with equivalent stretches of the polynucleotide of the invention even though the overall homology between the two polynucleotides may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.
A particularly preferred polynucleotide comprises the nucleotide sequence
(as given in FIG. 12, nucleotides 2872 to 5249) or a fragment or variant thereof.
This polynucleotide encodes HMPHP SCoA cleavage enzyme activity and a trans-feruloyl SCoA hydratase activity from Ps. fluorescens biovar. V, strain AN103 and encodes the preferred polypeptide of the fifth and sixth aspects of the invention.
A further particularly preferred polynucleotide comprises the nucleotide sequence
(as given in FIG. 12, nucleotides 3804 to 5249)
or a fragment or variant thereof. This polynucleotide encodes the polypeptide sequence of the sixth aspect of the invention. It is particularly convenient to isolate the whole gene from cosmid clone pFI 793 as deposited under the Budapest Treaty at NCIMB under Accession No NCIMB 40777.
The polynucleotides of the invention are all readily isolated from pFI 793 by probing with the given sequences or parts thereof, or by other methods known in the art, and the nucleotide sequences can be confirmed by reference to the deposited cosmid (pFI 793).
It will be appreciated that fragments and variants of the polynucleotides of the invention can readily be made by the person skilled in the art using standard molecular biological methods such as those described in Sambrook et al xe2x80x9cMolecular Cloning, a laboratory manualxe2x80x9d, (1989), (2nd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The whole gene and variants and fragments thereof are specifically included in this aspect of the invention.
It is preferred if the polynucleotide, conveniently DNA, is joined to a nucleic acid vector.
DNA constructs of the invention may be purified from the host cell using well known methods.
For example, plasmid vector DNA can be prepared on a large scale from cleaved lysates by banding in a CsCl gradient according to the methods of Clewell and Helinski (1970) Biochemistry 9, 4428-4440 and Clewell (1972) J. Bacteriol. 110, 667-676. Plasmid DNA extracted in this way can be freed from CsCl by dialysis against sterile, pyrogen-free buffer through Visking tubing or by size-exclusion chromatography.
Alternatively, plasmid DNA may be purified from cleared lysates using ion-exchange chromatography, for example those supplied by Qiagen (Chatsworth, Calif., USA). Hydroxyapatite column chromatography may also be used.
The DNA is then expressed in a suitable host to produce a polypeptide of the invention. Thus, a DNA encoding a polypeptide of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in U.S. Pat. Nos. 4,440,859 issued Apr. 3, 1984 to Rutter et al, 4,530,901 issued Jul. 23, 1985 to Weissman, 4,582,800 issued Apr. 15, 1986 to Crowl, 4,677,063 issued Jun. 30, 1987 to Mark et al, 4,678,751 issued Jul. 7, 1987 to Goeddel, 4,704,362 issued Nov. 3, 1987 to Itakura et al, 4,710,463 issued Dec. 1, 1987 to Murray, 4,757,006 issued Jul. 12, 1988 to Toole, Jr. et al, 4,766,075 issued Aug. 23, 1988 to Goeddel et al, and 4,810,648 issued Mar. 7, 1989 to Stalker, all of which are incorporated herein by reference.
The DNA encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the recombinant DNA of the invention may then be cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which, if desirable, can then be recovered.
Many expression systems are known, including bacteria (for example Escherichia coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells and whole plants, animal cells and insect cells.
The vectors include a prokaryotic replicon, such as the Co1E1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli transformed therewith.
Several promoters are available to direct transcription of bacterial and other heterologous genes in plants. In particular, these include the 35S promoter of cauliflower mosaic virus (CaMV 35S), the ribulose bisphosphate carboxylase small subunit promoter and the Agrobacterium T-DNA octopine synthase and manopine synthase promoters. These promoters have been widely used, for example, in conjunction with bacterial genes conferring herbicide resistance (see D. M. Stalker, ibid., pp 82-104). These promoters do not confer any specificity of gene expression at the organ, tissue or organellar levels, or responsiveness of gene expression to environmental influences such as light.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps)
A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3xe2x80x2-single-stranded termini with their 3xe2x80x2-5xe2x80x2-exonucleolytic activities, and fill in recessed 3xe2x80x2-ends with their polymerizing activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.
In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic and it may be comprised in a multicellular organism such as a plant. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeast and plant cells. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred plant host cells and plants include those from Nicotiana spp., Solanum tuberosum (potato), Brassica spp. (eg oil seed rape), Beta spp. (eg sugar beet, leaf beet and beetroot), Capsicum spp. and Vanilla spp.
Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to plant cells and whole plants three plant transformation approaches are typically used (J. Draper and R. Scott in D. Grierson (ed.), xe2x80x9cPlant Genetic Engineeringxe2x80x9d, Blackie, Glasgow and London, 1991, vol. 1, pp 38-81):
i) Agrobacterium-mediated transformation, using the Ti plasmid of A. tumefaciens and the Ri plasmid of A. rhizogenes (P. Armitage, R. Walden and J. Draper in J. Draper, R. Scott, P. Armitage and R. Walden (eds.), xe2x80x9cPlant Genetic Transformation and Expressionxe2x80x94A Laboratory Manualxe2x80x9d, Blackwell Scientific Publications, Oxford, 1988, pp 1-67; R. J. Draper, R. Scott and J. Hamill ibid., pp 69-160);
ii) DNA-mediated gene transfer, by polyethylene glycol-stimulated DNA uptake into protoplasts, by electroporation, or by microinjection of protoplasts or plant cells (J. Draper, R. Scott, A. Kumar and G. Dury, ibid., pp 161-198);
iii) transformation using particle bombardment (D. McCabe and P. Christou, Plant Cell Tiss. Org. Cult., 3, 227-236 (1993); P. Christou, Plant J., 3, 275-281 (1992)).
Agrobacterium-mediated transformation is generally ineffective for monocotyledonous plants (eg Vanilla), for which approaches ii) and iii) are therefore preferred. In all approaches a suitable selection marker, such as kanamycin- or herbicide-resistance, is preferred or alternatively a screenable marker (xe2x80x9creporterxe2x80x9d) gene, such as xcex2-glucuronidase or luciferase (see J. Draper and R. Scott in D. Grierson (ed.), xe2x80x9cPlant Genetic Engineeringxe2x80x9d, Blackie, Glasgow and London, 1991, vol. 1 pp 38-81).
Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cell, bacterial cells and plant cells.
For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5xc3x97PEB using 6250 V per cm at 25 xcexcFD.
Methods for transformation of yeast by electroporation are disclosed in Becker and Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.
In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium; and also, in the case of plant cells, a plant derived from, and containing, such cells.
It is particularly preferred if the host cell comprises a nucleic acid which encodes any one of, or combination of, a polypeptide which, in the presence of appropriate cofactors if any is capable of catalysing the interconversion of trans-feruloyl SCoA and 4-hydroxy-3-methoxyphenyl-xcex2-hydroxypropionyl SCoA (HMPHP SCoA) or a polypeptide which, in the presence of appropriate cofactors if any, is capable of catalysing the interconversion of 4-hydroxy-3-methoxyphenyl-xcex2-hydroxypropionyl SCoA (HMPHP SCoA) and vanillin.
It will be appreciated that the host cells of the invention or an extract thereof are particularly suited for use in the methods of the invention. It is particularly preferred if the host cell does not contain means for converting vanillin to a non-vanillin product.
It is most preferred if the host cell is a plant cell or is comprised in a whole plant or a bacterial cell or a yeast cell. Preferred bacterial hosts include lactic acid bacteria such as Lactococcus spp. and Lactobacillus nspp. Preferred yeast hosts include Saccharontyces cerevisiae and its biovars. It is particularly preferred if the host cell is a food-grade host cell (for example a microorganism which is used or can be used in the food or beverage industry). It is also preferred if the plant is an edible plant.
It will be appreciated that some host cells or host organisms may already contain enzyme activities I, II, III or IV and, in that case, it may be sufficient, in order to use the host cells in the methods of the invention, to introduce into said host cell or host organism one or more polynucleotides which encode enzyme activities II, III or IV which encode those enzyme activities which are deficient in the host cell or host organisms.
In the case of plants for use in the methods of the invention, it is preferred that the relevant gene expression is directed to target organs, tissues and subcellular organelles where trans-feruloyl SCoA, or other appropriate substrates (eg 4-trans-coumaroyl SCoA or trans-caffeoyl ScoA) for the enzymes encoded by the transferred genes, are most readily available. The stage at which thioesterification with CoASH occurs in plants, in relation to the progressive substitution of the phenyl ring which takes place during the conversion from trans-cinnamate to trans-ferulate, is unclear and may be variable (see R. Whetten and R. Sederoff, The Plant Cell, 7, 1001-1013 (1995)). The subcellular localisation or distribution of these intermediates during plant phenylpropanoid metabolism also remains uncertain; it is likely that they are cytosolic, or that some functional organisation of the enzymes which metabolise them occurs. The concept of the metabolic cluster or xe2x80x9cmetabolonxe2x80x9d, in which there is a degree of metabolic channelling and free diffusion is restricted, has been proposed and discussed (see R. A. Dixon and N. L. Paiva, The Plant Cell, 7, 1085-1097 (1995) and Ioc cit.).
Trans-ferulate, 4-trans-coumarate and trans-caffeate are normal metabolic intermediates. Thus, there may be no requirement to manipulate host plants in order to provide trans-feruloyl SCoA. Their concentrations are expected to be influenced in varying degrees by physiological requirements for a wide range of end-products of the phenylpropanoid pathway, including for example lignin, coumarins and flavonoids. There is some evidence that the activity of the first enzyme of the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL: EC 4.3.1.5), can influence the accumulation of end-products of the pathway (N. Bate, J. Orr, W. Ni, A. Meromi, T. Nadler-Hassar, P. W. Doerner, R. A. Dixon, C. J. Lamb and Y. Elkind, Proc. Natl. Acad. Sci. USA, 91, 7608-7612 (1994)) so under certain circumstances it is possible to enhance the metabolic effects of the expression of the genes for enzyme activities II, and III by increasing the expression of PAL.
It is well known that gene expression in the phenylpropanoid pathway is responsive to a range of environmental and stress factors, including wounding, chemical elicitors of pathogenic origin, and u/v light. The mechanisms regulating these responses are not very well understood, though several transcription factors have been identified (see R. A. Dixon and N. L. Paiva, The Plant Cell, 7, 1085-1097 (1995)). However, particularly when these are more fully characterised, they do or will offer opportunities for predictable and inducible control of gene expression, particular to enhance the provision of substrate.
Thus, a further aspect of the invention provides a transgenic plant comprising a polynucleotide according to any of the fourth or fifth aspects of the invention. In other words, the transgenic plant is genetically engineered to encode and, preferably, express any one or more of enzyme activities II or III. It is particularly preferred that, following said genetic engineering the plant is able to produce vanillin from trans-feruloyl SCoA. It will be appreciated that, depending on the enzymes present in the host plant, it may be necessary only to provide a gene encoding only a single of said enzyme activities or it may be necessary to provide a gene or genes of any two of said enzyme activities.
Conveniently, the transgenic plant is genetically engineered to encode and, preferably, express enzyme activities II and III. It can be readily seen that the transgenic plants of this aspect of the invention may be used in the methods of producing vanillin of the invention, especially when the plant provides the enzyme activity that interconverts trans-ferulic acid or a salt thereof and trans-feruloyl SCoA.
Preferably the plant is a plant which is readily transformed. Preferably the plant is a plant which is commonly used in agriculture or horticulture and more preferably the plant is an edible plant. Advantageously the plant is a plant in which it is desirable to introduce a vanilla flavour or aroma.
Preferred plants include those selected from Nicotiana spp., Solanum tuberosum, Brassica spp., Capsicum spp., Beta spp. and Vanilla spp.
As is described in more detail below the plant may be eaten or may be processed into a foodstuff or beverage. Thus, conveniently the transgenic plant is processed or prepared so that it is not capable of reproduction or cultivation, for example the transgenic plant is harvested from the environment in which it was grown.
When vanillin (or the desirable products such as p-hydroxybenzaldehyde) is produced in a host cell or organism of the invention, especially if it is produced in a transgenic plant of the invention, the vanillin or desirable product may initially be present in the form of a glycoside, more particularly, a xcex2-D-glycoside, or, in the case of a carboxylic acid, as esters of xcex2-D-glucose (as occurs in Vanilla pod). In this case, it is desirable to release the vanillin (and desirable product) into its uncombined form, for example by acid- or base-catalysed hydrolysis or by the use of glycosides such as the xcex2-D-glucosidase (emulsin; S. Hestrin, D. S. Femgold and M. Schramm, Meth. Enzymol. I, 231-257 (1955); see also D. Chassagne, C. Bayonore, J. Crouzet and K. Baumes in xe2x80x9cBioflavour 95xe2x80x9d, eds. P. Étixc3xa9vant and P. Schreier, INRA, Paris, pages 217-222 (1995).).
In relation to the use of a microorganism such as Ps. fluorescens biovar V, strain AN103 or of a microorganism which has been genetically modified to contain enzyme activities II and III (or at least those of these activities that it does not normally have), it is preferred that said microorganism is provided with trans-feruloyl SCoA or a means to provide said CoA thioester from trans-fernlic acid or a salt thereof at least in its culture medium.
Thus, it can be seen from all of the foregoing description that the invention includes biochemical and fermentative processes for producing vanillin and vanillic acid, recombinant or transgenic plants and the use of the said plants in a method of making vanillin or vanillic acid.
Typically, in a biochemical process the strain of Pseudomonas (eg Ps. fluorescens biovar. V, strain AN 103) provides an enzyme system for the biotransformation of plant derived trans-ferulic acid to vanillin and/or related compounds. Enzyme preparations, whole cells of Pseudomonas or a heterologous host organism expressing appropriate Pseudomonas genes may be used for this. A variety of mutants of Pseudomonas and various additional enzyme preparations, co-factors or co-factor regenerating systems may be used. The Pseudomonas enzymes might be overexpressed in a heterologous host before being extracted and used in a biotransformation.
Alternatively, but suitably, some form of fermentation process may be used which involves the Pseudomonas strain or an appropriate derived mutant or a heterologous host organism in which the genes for biotransformation are expressed. The chosen microorganism is typically grown on a ferulate-rich substrate or a substrate comprising trans-feruloyl SCoA. This could generate a vanillin production process.
In addition the invention includes recombinant microorganisms (eg lactic acid bacteria) which are modified to contain genes encoding enzyme activities II and III. For example, lactic acid bacteria modified according to the invention may be used to produce vanilla-flavoured yoghurt provided that they are supplied with trans-feruloyl SCoA.
Advantageously, genes for vanillin production (such as those encoding enzyme activities II and III) are expressed in a variety of plant species such that vanillin accumulates in an appropriate tissue. In this case a new crop plant may be cultivated and vanillin would be extracted. Thus, a sugar beet plant may be made according to the invention in which the beet was rich in vanillin. In addition the development of a novel plant cultivars for direct consumption (eg vanilla-flavoured capsicum), or even for their desirable aroma properties, included in the invention.
The polypeptides of the invention, or the genes which encode them, may be used either individually or in combination (whether as substantially isolated polypeptides, or in cell-free extracts or as host cells or organisms which encode and, preferably, express said polypeptides) to convert a compound into a desirable product. Certain compounds and desirable products have been described above. However, the invention also includes the production of any other desirable product, such as a flavour or aroma, from substrates related to trans-ferulate and other known substrates of the polypeptides (enzymes) of the invention. Thus, the polypeptides or genes of the invention, either individually or in combination, may be used in processes for converting, for example, synthetic substrates of the said polypeptides (enzymes) into novel flavours and aromas or they may be used to modify the chemical profile of known flavours or aromas.
Similarly, it will be appreciated that certain desirable products can be made by the further action of enzyme activity IV upon certain compounds, particularly those produced by enzyme activities I, II and III. For example, enzymes activities I, II and III may be used to convert trans-4-coumaric acid or trans4-coumaroyl SCoA to p-hydroxybenzaldehyde and enzyme activity IV may then be used to convert p-hydroxybenzaldehyde to p-hydroxy-benzoic acid. Thus, the invention includes a method of producing p-hydroxy-benzoic acid using at least one of enzyme activities I, II, III and IV and advantageously using all of them.
A further aspect of the invention provides a food or beverage comprising a host cell comprising a polynucleotide of the invention, or an extract of said host cell. The host cells comprising one or more polynucleotides of the invention, especially those such host cells which produce vanillin by virtue of the presence of said polynucleotide or polynucleotides, may be used in the production of food or beverages. In particular, as discussed above, lactic acid bacteria which produce vanillin by the methods of the invention may be used in the production of cheese, yogurt and related products including milk drinks. Similarly, yeasts which produce vanillin by the methods of the invention may be used in the production of food and beverages such as bread and beer. Extracts of said host cells may also be used in the food or beverage industry.
A still further aspect of the invention provides a food or beverage comprising a transgenic plant comprising a polynucleotide of the invention, or a part or extract or said transgenic plant. The transgenic plant comprising one or more polynucleotides of the invention, especially those such transgenic plants which produce vanillin by virtue of the presence of said polynucleotide or polynucleotides, may constitute the food itself or they may be processed to form the food or beverage. For example, a tuber of a transgenic potato of the invention constitutes a food of this aspect of the invention. Alternatively, said transgenic potato may be processed into another foodstuff which is, nevertheless, a food of this aspect of the invention.
Still further aspects of the invention provide use of Pseudomonas fluorescens biovar. V, strain AN103 or a mutant or derivative thereof in a method for producing vanillin, or vanillic acid or salt thereof; use of a polypeptide of the invention in a method for producmg vanillin, or vanillic acid or salt thereof; use of a polynucleotide of the invention in a method for producing vanillin, or vanillic acid or salt thereof; and use of a host cell of the invention in a method for producing vanillin or vanillic acid or a salt thereof.
As is clear from the foregoing the invention also includes a method of producing vanillin or vanillic acid, or other related products, the method comprising providing trans-feruloyl SCoA (or any other suitable CoASH thioester which can be acted upon by enzyme activity II) and providing enzyme activity II, enzyme activity III and, in the case of vanillic acid production or another related product, enzyme activity IV. Trans-feruloyl SCoA is obtainable by the method described in Example 2 or it may be obtained using the methods of Zenk et al (1980) Anal. Biochem. 101, 182-187, incorporated herein by reference. Other CoASH thioesters which may be substrates for enzyme II are also described in Zenk et al.
The preferred method steps and organisms for use in the methods, and the foods and beverages of the earlier aspects of the invention are also preferred in this method of the invention to the extent that they are compatible with this method, and the organisms used in this method.
As is discussed above, trans-feruloyl SCoA (and related CoASH thioesters such as 4-trans-coumaroyl SCoA and trans-caffeoyl SCoA) are normal metabolic intermediates in plants. Thus, a transgenic plant which comprises a polynucleotide or polynucleotides which encode, and preferably express, enzyme activities II and III in a location in the plant which contains trans-feruloyl SCoA or other suitable CoASH thioester is particularly suited for the purposes of this aspect of the invention. As is described above, such transgenic plants and products derived therefrom form part of the invention.
The invention will now be described in more detail with reference to the following Examples and Figures wherein: